Annals of Diagnostic Pathology 18 (2014) 74–77
Contents lists available at ScienceDirect
Annals of Diagnostic Pathology
Positive nuclear expression of KLF8 might be correlated with shorter survival in gastric adenocarcinoma☆,☆☆ Li-Sung Hsu, PhD a, b, 1, Pei-Ru Wu, MD c, 1, Ken-Tu Yeh, MD c, e, Chung-Min Yeh, MS c, Ko-Hung Shen, MD c, Chih-Jun Chen, MD c, d, e,⁎, Maw-Soan Soon, MD f,⁎ a
Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan d Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan e School of Medicine, Chung Shan Medical University, Taichung, Taiwan f Department of Gastroenterology, Changhua Christian Hospital, Changhua, Taiwan b c
a r t i c l e
i n f o
Keywords: Gastric adenocarcinoma Krűppel-like factor 8 survival
a b s t r a c t Krűppel-like factor 8 (KLF8) is important in cell proliferation, epithelial-to-mesenchymal transition, cell migration, and invasion. Gastric adenocarcinoma is among the leading causes of cancer-related death in the world. In this study, the clinicopathologic correlation of KLF8 expression with gastric adenocarcinoma in Taiwan was investigated. The nuclear localization of KLF8 was correlated with advanced stage (P = .008) and 3-year survival rate (P = .043). The nuclear expression of KLF8 was significantly higher in the diffused type of gastric adenocarcinoma compared with the intestinal type (P = .036). Kaplan-Meier analysis results showed that patients with positive nuclear KLF8 had significantly lower overall survival rate compared with those with negative nuclear KLF8 (P = .011). Univariate analysis results indicated that positive nuclear KLF8 expression, advanced stage, and lymph node metastasis are correlated with lower overall survival. Positive nuclear KLF8 might be correlated with lower survival in gastric adenocarcinoma patients and might be an oncogene property in gastric adenocarcinoma carcinogenesis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Krűppel-like factors (KLFs), originally identified from Drosophila, contain 3 C2H2 zinc finger motifs, which can bind to specific DNA with guanine-cytosine–rich or CACCC sequences [1]. Krűppel-like factors may function as a transcription activator or repressor to regulate cellular functions including cell proliferation, apoptosis, stem cell renewal, and tumorigenesis [1]. The function of an ubiquitously expressed KLF, which is known as KLF8 in carcinogenesis, was recently recognized [2]. Krűppel-like factor 8 was highly expressed in the cancerous part of renal cell carcinoma [3], glioma [4], and ovarian cancer [5]. Zhao et al [6] have
☆ This work was supported by grant 101-CCH-IRP-32 from Changhua Christian Hospital, Changhua, Taiwan. ☆☆ Conflict of interest: We declare that we did not have any conflict of interest. ⁎ Corresponding authors: C-J Chen, is to be contacted at Department of Surgical Pathology, M-S Soon, Department of Gastroenterology, Changhua Christain Hospital, No. 135 Nanxiao St, Changhua City, 500, Taiwan. Tel.: +886 4 7238595x4832; fax: +886 4 7232942. E-mail addresses: [email protected] (C.-J. Chen), [email protected] (M.-S. Soon). 1 These authors contributed equally to this work. 1092-9134/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.anndiagpath.2013.12.001
demonstrated that KLF8, a downstream target of focal adhesion kinase, increased cell cycle progression through the regulation of cyclin D expression. The overexpression of KLF8 in NIH3T3 cells triggered serum-independent growth and morphological transformation. Down-regulation of E-cadherin and increased invasion and migration were observed in human normal breast epithelial cell line MCF-10A cells that overexpressed KLF8 [7]. Inversed association of KLF8 and E-cadherin level was also detected in human breast cancer [7]. Knockdown of KLF8 by using small interference RNA (siRNA) eliminated migration, induced apoptosis, and decreased tumorigenesis of renal carcinoma 786-0 cells [3]. Similarly, KLF8 was involved in cell proliferation and migration in hepatocellular carcinomas [8]. High KLF8 expression was correlated with poor prognosis and early recurrence in hepatocellular carcinoma [8]. Wang et al [9] found that KLF8 stimulated the expression of matrix metalloproteinase 9, which in turn promoted migration and invasion in breast cancer. Knockdown of KLF8 expression by siRNAs suppressed cell growth and triggered apoptosis in glioblastoma cells [10]. These observations suggest the oncogenic function of KLF8 in cancer formation. Gastric cancer is among the leading causes of cancer-related death all over the world. We have previously demonstrated that the nuclear localization of KLF5 was correlated with advanced clinical stages, lymph node metastasis, and lower overall survival rate in gastric cancer patients [11]. Down-regulation of KLF8 by lentivirus-mediated
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chemical staining. Each tumor was given a score according to the intensity of the nuclear staining: no staining, 0; weak staining, 1+; moderate staining, 2+; and strong staining, 3+. Staining intensity was confirmed by 2 pathologists. The nuclear KLF8 expression was classified as either negative (b1) or positive (≥1).
2.4. Statistical analysis Statistical analysis was performed using the SPSS software (SPSS, Inc, Chicago, IL). The correlation between the expression of KLF8 and the different clinicopathologic parameters was measured by χ 2 and Fisher exact tests. The overall survival rate was determined by KaplanMeier plot and log-rank test. Multivariate analysis based on a Cox proportional hazard regression model was used to evaluate the prognostic significance of the clinical variables. P b .05 was considered to be statistically significant.
Fig. 1. Immunohistochemical staining for KLF8 expression in gastric carcinomas. Representative immunohistochemistry images of negative (left panel) and positive (right panel) nuclear KLF8 in gastric adenocarcinomas. Upper panel: magnification ×100. Lower panel: magnification ×400.
siRNA infection not only inhibited cell growth but also blocked cell invasion of gastric cancer cells [12,13]. In the current study, KLF8 expression and the clinicopathlogic parameters of gastric adenocarcinoma patients in Taiwan were investigated. 2. Materials and methods 2.1. Sample collection A total of 73 gastric cancer tissue samples from tissue blocks were enrolled in this study. The samples were staged and graded according to the TNM Classification of Malignant Tumors and the World Health Organization classification system. All the clinical parameters, including age, sex, tumor size, lymph node status, grade, stage, and overall survival, were obtained. 2.2. Tissue microarrays One tissue core with a diameter of 2 mm was obtained from each formalin-fixed, paraffin-embedded block of cancer and noncancer tissues. The tissue cores were arranged into new paraffin blocks by using a fine steel needle to create the tissue microarrays. CJ Chen and PR Wu examined a 4-μm hematoxylin and eosin–stained section to confirm the presence of the original cancers as indicated by morphologically representative areas. 2.3. Analyses of KLF8 expression by immunohistochemistry All immunohistochemistry procedures were performed as previously described [11]. The tissue microarray sections were deparaffinized, treated with 3% H2O2 in methanol, hydrated with gradient alcohol and phosphate-buffered saline solution, and incubated in 10 mmol/L citrate buffer. The antigen was retrieved by heating at 100°C for 20 minutes in 10 mmol/L citrate buffer (pH 6.0). After incubation with anti-KLF8 antibody (Sigma-Aldrich, St. Louis, MO) for 20 minutes at room temperature, the slides were incubated with a horseradish peroxidase/Fab polymer conjugate for an additional 30 minutes and then washed 3 times with phosphate-buffered saline solution. The sites of peroxidase activity were developed using 3,3′-diaminobenzidine tetrahydrochloride as a substrate and counterstained with hematoxylin. Positive and negative control samples were also included in the immunohisto-
3. Results 3.1. Patient characteristics A total of 73 gastric adenocarcinoma samples, including 22 females aged from 41 to 83 (mean±SD: 64.75±13.05) and 51 males aged from 42 to 89 (mean±SD: 67.26±11.02), were included in this study. A total of 19 cases were classified as T1 and T2 patients and 54 as T3 and T4 patients. A total of 26 cases did not show lymph node metastasis, whereas 47 cases showed lymph node metastasis. Distance metastasis was found only in 2 cases. A total of 54 cases were in the low clinical stage (stage I, II, and III) and 19 in the advanced clinical stage (stage IV). A total of 27 diffused type and 46 intestinal type gastric adenocarcinomas were included. Table 1 Correlation of nuclear expression of KLF8 with clinicopathologic parameters in gastric adenocarcinomas Nuclear staining of KLF8
Total
P
Negative
Positive
Sex F M
10 (45.5) 12 (23.5)
12 (54.5) 39 (76.5)
22 51
.094
Grade Well, moderate Poor
12 (36.4) 10 (25.0)
21 (63.6) 30 (75.0)
33 40
.317
T status T1,T2 T3,T4
9 (47.4) 13 (24.1)
10 (52.6) 41 (75.9)
19 54
.081
Lymph node metastasis No Yes
9 (34.6) 13 (27.7)
17 (65.4) 34 (72.3)
26 47
.599
Distance metastasis No Yes
22 (31.0) 0 (0)
49 (69.0) 2 (100)
71 2
1.000
Stage I, II, III IV
21 (38.9) 1 (5.3)
33 (61.1) 18 (94.7)
54 19
.008⁎
Survival ≤3 y N3 y
7 (18.9) 15 (41.70
30 (81.1) 21 (58.3)
37 36
.043⁎
Type D I
4 (14.8) 18 (39.1)
23 (85.2) 28 (60.9)
27 46
.036⁎
Abbreviations: F, female; M, male. P value was measured by Fisher exact test. ⁎ P b .05.
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Fig. 2. A Kaplan-Meier survival curve for the 73 gastric cancer patients with nuclear expression of KLF8 protein. Patients with positive nuclear staining for KLF8 had a significantly lower overall survival rate compared with patients with negative nuclear staining, as defined by a log-rank test (P = .011).
3.2. Correlation of KLF8 expression with clinicopathologic parameters in gastric adenocarcinoma The expression of KLF8 was assessed by immunohistochemical staining, in which an antibody was used against KLF8. Krűppel-like factor 8 expression level was scored as positive or negative according to the nuclear expression intensity (Fig. 1). As shown in Table 1, the nuclear expression of KLF8 was not correlated with sex (P = .094), grade (P = .317), T status (P = .081), lymph node metastasis (P = .599), and distance metastasis (P = 1.000). A significantly higher nuclear expression of KLF8 was found in advanced clinical stages (P = .008), lower 3-year survival rates (P = .043), and diffused type of gastric adenocarcinoma (P = .036) (Table 1). 3.3. Survival analysis Kaplan-Meier survival analysis and log-rank test were conducted to compare the survival rate of gastric adenocarcinoma with different expressions of nuclear KLF8. Patients with negative nuclear staining displayed a significantly higher overall survival rate compared with those with positive nuclear staining (Fig. 2). Positive nuclear stain of KLF8 (95% confidence intervals [CI], 1.25.2; P = .014), stage (95% CI, 2.2-7.6; P b .001), and lymph node metastasis (95% CI, 1.2-4.5; P = .013) affected the overall survival, as shown by univariate analysis results (Table 2). However, multivariate Cox proportional hazard analysis indicated that only the advanced clinical stage was the independent factor for the overall survival of gastric adenocarcinomas (95% CI, 1.2-5.5; P = .011) (Table 3). 4. Discussion Kruppel-like family proteins are important in cell proliferation, apoptosis, and differentiation [1]. Krűppel-like factor 8, which was originally identified as a downstream target of focal adhesion kinase, can transform and up-regulate in several human cancer cell lines
[14,15]. The association between the KLF8 expression and clinical parameters of gastric adenocarcinomas was demonstrated. Based on our immunohistochemical analysis, the positive nuclear expression of KLF8 was correlated with advanced tumor stage and lower overall survival rate of gastric adenocarcinoma. Aberrant overexpression of KLF8 was found in several human cancers in previous reports [2]. Significantly higher expression of KLF8 was observed in malignant and metastatic ovarian cancers compared with benign and normal ovarian regions [5]. A stable expression of KLF8 in T80 cells could cause tumor and reduce the life span in a xenograft model [5]. High nuclear expression of KLF8 was an independent prognosis and recurrence factor in hepatocellular carcinomas [8]. Ectopic expression of KLF8 increased cell proliferation and invasion and altered the epithelial-to-mesenchymal transition [8]. Furthermore, KLF8 expression was elevated in renal cell carcinomas compared with those in
Table 2 Univariate analysis of overall survival rate in gastric adenocarcinomas Univariate survival analysis Variable
Hazard ratio
95% CI
KLF8 nuclear stain No Yes
1.0 2.5
1.2-5.2
.014⁎
Grade Well/moderate Poor
1.0 1.4
0.8-2.5
.232
Stage I, II, III IV
1.0 4.1
2.2-7.6
b.001⁎
Lymph node metastasis No Yes
1.0 2.3
1.2-4.5
.013⁎
⁎ P b 0.05.
P
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References
Table 3 Multivariate analysis of overall survival rate in gastric adenocarcinomas Multivariate survival analysis Variable
Hazard ratio
95% CI
P
KLF8 nuclear stain No Yes
1.0 1.9
0.8-4.1
.123
Grade Well/moderate Poor
1.0 1.2
0.7-2.2
.469
Stage I, II, III IV
1.0 2.6
1.2-5.5
.011
Lymph node metastasis No Yes
1.0 1.6
0.8-3.4
.220
adjacent nontumor parts [3]. Knockdown of KLF8 by siRNA inhibited cell growth and invasion [3]. Liu et al [13] have shown that higher KLF8 expression was observed in gastric cancer parts and cancer cell lines compared with normal gastric tissue and mucosa cells. The downregulation of KLF8 by lentivirus-delivered siRNA reduced cell proliferation, triggered apoptosis, and reduced tumorigenesis of SCG7901 cells [13]. In addition, blocked KLF8 expression also controlled the invasion and transformation of the growth factor β1–induced epithelial-tomesenchymal transition of gastric cancer cells [12,16]. Thus, KLF8 is important in gastric cancer proliferation. In this study, we demonstrated that nuclear localization of KLF8 was significantly higher in the diffused type gastric cancer than in the intestinal type (P = .036). These 2 types of gastric cancer arise from distinct genetic alternations. HER2/neu amplification or overexpression was found in 10% to 15% of the intestinal type but not in the diffused type [17]. By contrast, loss of E-cadherin expression was detected in 50% diffused type but not in intestinal type gastric adenocarcinoma [18,19]. Overexpressions of urokinase plasminogen activator, cyclooxygenase 2, and vascular endothelial growth factor were predominantly found in intestinal type gastric adenocarcinomas [20-22]. E-cadherin, which can be repressed by KLF8, was downregulated in the diffused type [18,19]. Krűppel-like factor 8 may cooperate with other proteins that are involved in the different types of gastric adenocarcinoma formation. In summary, positive nuclear expression of KLF8 was correlated with advanced clinical stage and diffused type. Kaplan-Meier analysis also indicated that positive nuclear staining of KLF8 was correlated with lower overall survival. However, KLF8 was not an independent prognostic factor. Krűppel-like factor 8 may have an oncogenic function in the carcinogenesis of gastric adenocarcinoma.
[1] McConnell BB, Yang VW. Mammalian Kruppel-like factors in health and diseases. Physiol Rev 2010;90:1337–81. [2] Lahiri SK, Zhao J. Kruppel-like factor 8 emerges as an important regulator of cancer. Am J Transl Res 2012;4:357–63. [3] Fu WJ, Li JC, Wu XY, Yang ZB, Mo ZN, Huang JW, et al. Small interference RNA targeting Kruppel-like factor 8 inhibits the renal carcinoma 786-0 cells growth in vitro and in vivo. J Cancer Res Clin Oncol 2010;136:1255–65. [4] Schnell O, Romagna A, Jaehnert I, Albrecht V, Eigenbrod S, Juerchott K, et al. Kruppel-like factor 8 (KLF8) is expressed in gliomas of different WHO grades and is essential for tumor cell proliferation. PLoS One 2012;7:e30429. [5] Lu H, Wang X, Urvalek AM, Li T, Xie H, Yu L, et al. Transformation of human ovarian surface epithelial cells by Kruppel-like factor 8. Oncogene 2012. http://dx.doi.org/ 10.1038/onc.2012.545. [6] Zhao J, Bian ZC, Yee K, Chen BP, Chien S, Guan JL. Identification of transcription factor KLF8 as a downstream target of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression. Mol Cell 2003;11:1503–15. [7] Wang X, Zheng M, Liu G, Xia W, McKeown-Longo PJ, Hung MC, et al. Kruppel-like factor 8 induces epithelial to mesenchymal transition and epithelial cell invasion. Cancer Res 2007;67:7184–93. [8] Li JC, Yang XR, Sun HX, Xu Y, Zhou J, Qiu SJ, et al. Up-regulation of Kruppel-like factor 8 promotes tumor invasion and indicates poor prognosis for hepatocellular carcinoma. Gastroenterology 2010;139:2146–57 [e12]. [9] Wang X, Lu H, Urvalek AM, Li T, Yu L, Lamar J, et al. KLF8 promotes human breast cancer cell invasion and metastasis by transcriptional activation of MMP9. Oncogene 2011;30:1901–11. [10] Wan W, Zhu J, Sun X, Tang W. Small interfering RNA targeting Kruppel-like factor 8 inhibits U251 glioblastoma cell growth by inducing apoptosis. Mol Med Rep 2012;5:347–50. [11] Soon MS, Hsu LS, Chen CJ, Chu PY, Liou JH, Lin SH, et al. Expression of Kruppel-like factor 5 in gastric cancer and its clinical correlation in Taiwan. Virchows Arch 2010;459:161–6. [12] Chen G, Yang W, Jin W, Wang Y, Tao C, Yu Z, et al. Lentivirus-mediated gene silencing of KLF8 reduced the proliferation and invasion of gastric cancer cells. Mol Biol Rep 2012;39:9809–15. [13] Liu L, Liu N, Xu M, Liu Y, Min J, Pang H, et al. Lentivirus-delivered Kruppel-like factor 8 small interfering RNA inhibits gastric cancer cell growth in vitro and in vivo. Tumour Biol 2012;33:53–61. [14] Lu H, Hu L, Li T, Lahiri S, Shen C, Wason MS, et al. A novel role of Kruppel-like factor 8 in DNA repair in breast cancer cells. J Biol Chem 2012;287:43720–9. [15] Wang X, Zhao J. KLF8 transcription factor participates in oncogenic transformation. Oncogene 2007;26:456–61. [16] Zhang H, Liu L, Wang Y, Zhao G, Xie R, Liu C, et al. KLF8 involves in TGF-beta– induced EMT and promotes invasion and migration in gastric cancer cells. J Cancer Res Clin Oncol 2013;139:1033–42. [17] Zhang XL, Yang YS, Xu DP, Qu JH, Guo MZ, Gong Y, et al. Comparative study on overexpression of HER2/neu and HER3 in gastric cancer. World J Surg 2009;33: 2112–8. [18] Chan AO, Chu KM, Lam SK, Wong BC, Kwok KF, Law S, et al. Soluble E-cadherin is an independent pretherapeutic factor for long-term survival in gastric cancer. J Clin Oncol 2003;21:2288–93. [19] Chan AO, Lam SK, Chu KM, Lam CM, Kwok E, Leung SY, et al. Soluble E-cadherin is a valid prognostic marker in gastric carcinoma. Gut 2001;48:808–11. [20] Beyer BC, Heiss MM, Simon EH, Gruetzner KU, Babic R, Jauch KW, et al. Urokinase system expression in gastric carcinoma: prognostic impact in an independent patient series and first evidence of predictive value in preoperative biopsy and intestinal metaplasia specimens. Cancer 2006;106:1026–35. [21] Han SL, Tang HJ, Hua YW, Ji SQ, Lin DX. Expression of COX-2 in stomach cancers and its relation to their biological features. Dig Surg 2003;20:107–14. [22] Vidal O, Metges JP, Elizalde I, Valentini M, Volant A, Molina R, et al. High preoperative serum vascular endothelial growth factor levels predict poor clinical outcome after curative resection of gastric cancer. Br J Surg 2009;96:1443–51.
Contents lists available at ScienceDirect
Annals of Diagnostic Pathology
Positive nuclear expression of KLF8 might be correlated with shorter survival in gastric adenocarcinoma☆,☆☆ Li-Sung Hsu, PhD a, b, 1, Pei-Ru Wu, MD c, 1, Ken-Tu Yeh, MD c, e, Chung-Min Yeh, MS c, Ko-Hung Shen, MD c, Chih-Jun Chen, MD c, d, e,⁎, Maw-Soan Soon, MD f,⁎ a
Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan d Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan e School of Medicine, Chung Shan Medical University, Taichung, Taiwan f Department of Gastroenterology, Changhua Christian Hospital, Changhua, Taiwan b c
a r t i c l e
i n f o
Keywords: Gastric adenocarcinoma Krűppel-like factor 8 survival
a b s t r a c t Krűppel-like factor 8 (KLF8) is important in cell proliferation, epithelial-to-mesenchymal transition, cell migration, and invasion. Gastric adenocarcinoma is among the leading causes of cancer-related death in the world. In this study, the clinicopathologic correlation of KLF8 expression with gastric adenocarcinoma in Taiwan was investigated. The nuclear localization of KLF8 was correlated with advanced stage (P = .008) and 3-year survival rate (P = .043). The nuclear expression of KLF8 was significantly higher in the diffused type of gastric adenocarcinoma compared with the intestinal type (P = .036). Kaplan-Meier analysis results showed that patients with positive nuclear KLF8 had significantly lower overall survival rate compared with those with negative nuclear KLF8 (P = .011). Univariate analysis results indicated that positive nuclear KLF8 expression, advanced stage, and lymph node metastasis are correlated with lower overall survival. Positive nuclear KLF8 might be correlated with lower survival in gastric adenocarcinoma patients and might be an oncogene property in gastric adenocarcinoma carcinogenesis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Krűppel-like factors (KLFs), originally identified from Drosophila, contain 3 C2H2 zinc finger motifs, which can bind to specific DNA with guanine-cytosine–rich or CACCC sequences [1]. Krűppel-like factors may function as a transcription activator or repressor to regulate cellular functions including cell proliferation, apoptosis, stem cell renewal, and tumorigenesis [1]. The function of an ubiquitously expressed KLF, which is known as KLF8 in carcinogenesis, was recently recognized [2]. Krűppel-like factor 8 was highly expressed in the cancerous part of renal cell carcinoma [3], glioma [4], and ovarian cancer [5]. Zhao et al [6] have
☆ This work was supported by grant 101-CCH-IRP-32 from Changhua Christian Hospital, Changhua, Taiwan. ☆☆ Conflict of interest: We declare that we did not have any conflict of interest. ⁎ Corresponding authors: C-J Chen, is to be contacted at Department of Surgical Pathology, M-S Soon, Department of Gastroenterology, Changhua Christain Hospital, No. 135 Nanxiao St, Changhua City, 500, Taiwan. Tel.: +886 4 7238595x4832; fax: +886 4 7232942. E-mail addresses: [email protected] (C.-J. Chen), [email protected] (M.-S. Soon). 1 These authors contributed equally to this work. 1092-9134/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.anndiagpath.2013.12.001
demonstrated that KLF8, a downstream target of focal adhesion kinase, increased cell cycle progression through the regulation of cyclin D expression. The overexpression of KLF8 in NIH3T3 cells triggered serum-independent growth and morphological transformation. Down-regulation of E-cadherin and increased invasion and migration were observed in human normal breast epithelial cell line MCF-10A cells that overexpressed KLF8 [7]. Inversed association of KLF8 and E-cadherin level was also detected in human breast cancer [7]. Knockdown of KLF8 by using small interference RNA (siRNA) eliminated migration, induced apoptosis, and decreased tumorigenesis of renal carcinoma 786-0 cells [3]. Similarly, KLF8 was involved in cell proliferation and migration in hepatocellular carcinomas [8]. High KLF8 expression was correlated with poor prognosis and early recurrence in hepatocellular carcinoma [8]. Wang et al [9] found that KLF8 stimulated the expression of matrix metalloproteinase 9, which in turn promoted migration and invasion in breast cancer. Knockdown of KLF8 expression by siRNAs suppressed cell growth and triggered apoptosis in glioblastoma cells [10]. These observations suggest the oncogenic function of KLF8 in cancer formation. Gastric cancer is among the leading causes of cancer-related death all over the world. We have previously demonstrated that the nuclear localization of KLF5 was correlated with advanced clinical stages, lymph node metastasis, and lower overall survival rate in gastric cancer patients [11]. Down-regulation of KLF8 by lentivirus-mediated
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chemical staining. Each tumor was given a score according to the intensity of the nuclear staining: no staining, 0; weak staining, 1+; moderate staining, 2+; and strong staining, 3+. Staining intensity was confirmed by 2 pathologists. The nuclear KLF8 expression was classified as either negative (b1) or positive (≥1).
2.4. Statistical analysis Statistical analysis was performed using the SPSS software (SPSS, Inc, Chicago, IL). The correlation between the expression of KLF8 and the different clinicopathologic parameters was measured by χ 2 and Fisher exact tests. The overall survival rate was determined by KaplanMeier plot and log-rank test. Multivariate analysis based on a Cox proportional hazard regression model was used to evaluate the prognostic significance of the clinical variables. P b .05 was considered to be statistically significant.
Fig. 1. Immunohistochemical staining for KLF8 expression in gastric carcinomas. Representative immunohistochemistry images of negative (left panel) and positive (right panel) nuclear KLF8 in gastric adenocarcinomas. Upper panel: magnification ×100. Lower panel: magnification ×400.
siRNA infection not only inhibited cell growth but also blocked cell invasion of gastric cancer cells [12,13]. In the current study, KLF8 expression and the clinicopathlogic parameters of gastric adenocarcinoma patients in Taiwan were investigated. 2. Materials and methods 2.1. Sample collection A total of 73 gastric cancer tissue samples from tissue blocks were enrolled in this study. The samples were staged and graded according to the TNM Classification of Malignant Tumors and the World Health Organization classification system. All the clinical parameters, including age, sex, tumor size, lymph node status, grade, stage, and overall survival, were obtained. 2.2. Tissue microarrays One tissue core with a diameter of 2 mm was obtained from each formalin-fixed, paraffin-embedded block of cancer and noncancer tissues. The tissue cores were arranged into new paraffin blocks by using a fine steel needle to create the tissue microarrays. CJ Chen and PR Wu examined a 4-μm hematoxylin and eosin–stained section to confirm the presence of the original cancers as indicated by morphologically representative areas. 2.3. Analyses of KLF8 expression by immunohistochemistry All immunohistochemistry procedures were performed as previously described [11]. The tissue microarray sections were deparaffinized, treated with 3% H2O2 in methanol, hydrated with gradient alcohol and phosphate-buffered saline solution, and incubated in 10 mmol/L citrate buffer. The antigen was retrieved by heating at 100°C for 20 minutes in 10 mmol/L citrate buffer (pH 6.0). After incubation with anti-KLF8 antibody (Sigma-Aldrich, St. Louis, MO) for 20 minutes at room temperature, the slides were incubated with a horseradish peroxidase/Fab polymer conjugate for an additional 30 minutes and then washed 3 times with phosphate-buffered saline solution. The sites of peroxidase activity were developed using 3,3′-diaminobenzidine tetrahydrochloride as a substrate and counterstained with hematoxylin. Positive and negative control samples were also included in the immunohisto-
3. Results 3.1. Patient characteristics A total of 73 gastric adenocarcinoma samples, including 22 females aged from 41 to 83 (mean±SD: 64.75±13.05) and 51 males aged from 42 to 89 (mean±SD: 67.26±11.02), were included in this study. A total of 19 cases were classified as T1 and T2 patients and 54 as T3 and T4 patients. A total of 26 cases did not show lymph node metastasis, whereas 47 cases showed lymph node metastasis. Distance metastasis was found only in 2 cases. A total of 54 cases were in the low clinical stage (stage I, II, and III) and 19 in the advanced clinical stage (stage IV). A total of 27 diffused type and 46 intestinal type gastric adenocarcinomas were included. Table 1 Correlation of nuclear expression of KLF8 with clinicopathologic parameters in gastric adenocarcinomas Nuclear staining of KLF8
Total
P
Negative
Positive
Sex F M
10 (45.5) 12 (23.5)
12 (54.5) 39 (76.5)
22 51
.094
Grade Well, moderate Poor
12 (36.4) 10 (25.0)
21 (63.6) 30 (75.0)
33 40
.317
T status T1,T2 T3,T4
9 (47.4) 13 (24.1)
10 (52.6) 41 (75.9)
19 54
.081
Lymph node metastasis No Yes
9 (34.6) 13 (27.7)
17 (65.4) 34 (72.3)
26 47
.599
Distance metastasis No Yes
22 (31.0) 0 (0)
49 (69.0) 2 (100)
71 2
1.000
Stage I, II, III IV
21 (38.9) 1 (5.3)
33 (61.1) 18 (94.7)
54 19
.008⁎
Survival ≤3 y N3 y
7 (18.9) 15 (41.70
30 (81.1) 21 (58.3)
37 36
.043⁎
Type D I
4 (14.8) 18 (39.1)
23 (85.2) 28 (60.9)
27 46
.036⁎
Abbreviations: F, female; M, male. P value was measured by Fisher exact test. ⁎ P b .05.
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Fig. 2. A Kaplan-Meier survival curve for the 73 gastric cancer patients with nuclear expression of KLF8 protein. Patients with positive nuclear staining for KLF8 had a significantly lower overall survival rate compared with patients with negative nuclear staining, as defined by a log-rank test (P = .011).
3.2. Correlation of KLF8 expression with clinicopathologic parameters in gastric adenocarcinoma The expression of KLF8 was assessed by immunohistochemical staining, in which an antibody was used against KLF8. Krűppel-like factor 8 expression level was scored as positive or negative according to the nuclear expression intensity (Fig. 1). As shown in Table 1, the nuclear expression of KLF8 was not correlated with sex (P = .094), grade (P = .317), T status (P = .081), lymph node metastasis (P = .599), and distance metastasis (P = 1.000). A significantly higher nuclear expression of KLF8 was found in advanced clinical stages (P = .008), lower 3-year survival rates (P = .043), and diffused type of gastric adenocarcinoma (P = .036) (Table 1). 3.3. Survival analysis Kaplan-Meier survival analysis and log-rank test were conducted to compare the survival rate of gastric adenocarcinoma with different expressions of nuclear KLF8. Patients with negative nuclear staining displayed a significantly higher overall survival rate compared with those with positive nuclear staining (Fig. 2). Positive nuclear stain of KLF8 (95% confidence intervals [CI], 1.25.2; P = .014), stage (95% CI, 2.2-7.6; P b .001), and lymph node metastasis (95% CI, 1.2-4.5; P = .013) affected the overall survival, as shown by univariate analysis results (Table 2). However, multivariate Cox proportional hazard analysis indicated that only the advanced clinical stage was the independent factor for the overall survival of gastric adenocarcinomas (95% CI, 1.2-5.5; P = .011) (Table 3). 4. Discussion Kruppel-like family proteins are important in cell proliferation, apoptosis, and differentiation [1]. Krűppel-like factor 8, which was originally identified as a downstream target of focal adhesion kinase, can transform and up-regulate in several human cancer cell lines
[14,15]. The association between the KLF8 expression and clinical parameters of gastric adenocarcinomas was demonstrated. Based on our immunohistochemical analysis, the positive nuclear expression of KLF8 was correlated with advanced tumor stage and lower overall survival rate of gastric adenocarcinoma. Aberrant overexpression of KLF8 was found in several human cancers in previous reports [2]. Significantly higher expression of KLF8 was observed in malignant and metastatic ovarian cancers compared with benign and normal ovarian regions [5]. A stable expression of KLF8 in T80 cells could cause tumor and reduce the life span in a xenograft model [5]. High nuclear expression of KLF8 was an independent prognosis and recurrence factor in hepatocellular carcinomas [8]. Ectopic expression of KLF8 increased cell proliferation and invasion and altered the epithelial-to-mesenchymal transition [8]. Furthermore, KLF8 expression was elevated in renal cell carcinomas compared with those in
Table 2 Univariate analysis of overall survival rate in gastric adenocarcinomas Univariate survival analysis Variable
Hazard ratio
95% CI
KLF8 nuclear stain No Yes
1.0 2.5
1.2-5.2
.014⁎
Grade Well/moderate Poor
1.0 1.4
0.8-2.5
.232
Stage I, II, III IV
1.0 4.1
2.2-7.6
b.001⁎
Lymph node metastasis No Yes
1.0 2.3
1.2-4.5
.013⁎
⁎ P b 0.05.
P
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References
Table 3 Multivariate analysis of overall survival rate in gastric adenocarcinomas Multivariate survival analysis Variable
Hazard ratio
95% CI
P
KLF8 nuclear stain No Yes
1.0 1.9
0.8-4.1
.123
Grade Well/moderate Poor
1.0 1.2
0.7-2.2
.469
Stage I, II, III IV
1.0 2.6
1.2-5.5
.011
Lymph node metastasis No Yes
1.0 1.6
0.8-3.4
.220
adjacent nontumor parts [3]. Knockdown of KLF8 by siRNA inhibited cell growth and invasion [3]. Liu et al [13] have shown that higher KLF8 expression was observed in gastric cancer parts and cancer cell lines compared with normal gastric tissue and mucosa cells. The downregulation of KLF8 by lentivirus-delivered siRNA reduced cell proliferation, triggered apoptosis, and reduced tumorigenesis of SCG7901 cells [13]. In addition, blocked KLF8 expression also controlled the invasion and transformation of the growth factor β1–induced epithelial-tomesenchymal transition of gastric cancer cells [12,16]. Thus, KLF8 is important in gastric cancer proliferation. In this study, we demonstrated that nuclear localization of KLF8 was significantly higher in the diffused type gastric cancer than in the intestinal type (P = .036). These 2 types of gastric cancer arise from distinct genetic alternations. HER2/neu amplification or overexpression was found in 10% to 15% of the intestinal type but not in the diffused type [17]. By contrast, loss of E-cadherin expression was detected in 50% diffused type but not in intestinal type gastric adenocarcinoma [18,19]. Overexpressions of urokinase plasminogen activator, cyclooxygenase 2, and vascular endothelial growth factor were predominantly found in intestinal type gastric adenocarcinomas [20-22]. E-cadherin, which can be repressed by KLF8, was downregulated in the diffused type [18,19]. Krűppel-like factor 8 may cooperate with other proteins that are involved in the different types of gastric adenocarcinoma formation. In summary, positive nuclear expression of KLF8 was correlated with advanced clinical stage and diffused type. Kaplan-Meier analysis also indicated that positive nuclear staining of KLF8 was correlated with lower overall survival. However, KLF8 was not an independent prognostic factor. Krűppel-like factor 8 may have an oncogenic function in the carcinogenesis of gastric adenocarcinoma.
[1] McConnell BB, Yang VW. Mammalian Kruppel-like factors in health and diseases. Physiol Rev 2010;90:1337–81. [2] Lahiri SK, Zhao J. Kruppel-like factor 8 emerges as an important regulator of cancer. Am J Transl Res 2012;4:357–63. [3] Fu WJ, Li JC, Wu XY, Yang ZB, Mo ZN, Huang JW, et al. Small interference RNA targeting Kruppel-like factor 8 inhibits the renal carcinoma 786-0 cells growth in vitro and in vivo. J Cancer Res Clin Oncol 2010;136:1255–65. [4] Schnell O, Romagna A, Jaehnert I, Albrecht V, Eigenbrod S, Juerchott K, et al. Kruppel-like factor 8 (KLF8) is expressed in gliomas of different WHO grades and is essential for tumor cell proliferation. PLoS One 2012;7:e30429. [5] Lu H, Wang X, Urvalek AM, Li T, Xie H, Yu L, et al. Transformation of human ovarian surface epithelial cells by Kruppel-like factor 8. Oncogene 2012. http://dx.doi.org/ 10.1038/onc.2012.545. [6] Zhao J, Bian ZC, Yee K, Chen BP, Chien S, Guan JL. Identification of transcription factor KLF8 as a downstream target of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression. Mol Cell 2003;11:1503–15. [7] Wang X, Zheng M, Liu G, Xia W, McKeown-Longo PJ, Hung MC, et al. Kruppel-like factor 8 induces epithelial to mesenchymal transition and epithelial cell invasion. Cancer Res 2007;67:7184–93. [8] Li JC, Yang XR, Sun HX, Xu Y, Zhou J, Qiu SJ, et al. Up-regulation of Kruppel-like factor 8 promotes tumor invasion and indicates poor prognosis for hepatocellular carcinoma. Gastroenterology 2010;139:2146–57 [e12]. [9] Wang X, Lu H, Urvalek AM, Li T, Yu L, Lamar J, et al. KLF8 promotes human breast cancer cell invasion and metastasis by transcriptional activation of MMP9. Oncogene 2011;30:1901–11. [10] Wan W, Zhu J, Sun X, Tang W. Small interfering RNA targeting Kruppel-like factor 8 inhibits U251 glioblastoma cell growth by inducing apoptosis. Mol Med Rep 2012;5:347–50. [11] Soon MS, Hsu LS, Chen CJ, Chu PY, Liou JH, Lin SH, et al. Expression of Kruppel-like factor 5 in gastric cancer and its clinical correlation in Taiwan. Virchows Arch 2010;459:161–6. [12] Chen G, Yang W, Jin W, Wang Y, Tao C, Yu Z, et al. Lentivirus-mediated gene silencing of KLF8 reduced the proliferation and invasion of gastric cancer cells. Mol Biol Rep 2012;39:9809–15. [13] Liu L, Liu N, Xu M, Liu Y, Min J, Pang H, et al. Lentivirus-delivered Kruppel-like factor 8 small interfering RNA inhibits gastric cancer cell growth in vitro and in vivo. Tumour Biol 2012;33:53–61. [14] Lu H, Hu L, Li T, Lahiri S, Shen C, Wason MS, et al. A novel role of Kruppel-like factor 8 in DNA repair in breast cancer cells. J Biol Chem 2012;287:43720–9. [15] Wang X, Zhao J. KLF8 transcription factor participates in oncogenic transformation. Oncogene 2007;26:456–61. [16] Zhang H, Liu L, Wang Y, Zhao G, Xie R, Liu C, et al. KLF8 involves in TGF-beta– induced EMT and promotes invasion and migration in gastric cancer cells. J Cancer Res Clin Oncol 2013;139:1033–42. [17] Zhang XL, Yang YS, Xu DP, Qu JH, Guo MZ, Gong Y, et al. Comparative study on overexpression of HER2/neu and HER3 in gastric cancer. World J Surg 2009;33: 2112–8. [18] Chan AO, Chu KM, Lam SK, Wong BC, Kwok KF, Law S, et al. Soluble E-cadherin is an independent pretherapeutic factor for long-term survival in gastric cancer. J Clin Oncol 2003;21:2288–93. [19] Chan AO, Lam SK, Chu KM, Lam CM, Kwok E, Leung SY, et al. Soluble E-cadherin is a valid prognostic marker in gastric carcinoma. Gut 2001;48:808–11. [20] Beyer BC, Heiss MM, Simon EH, Gruetzner KU, Babic R, Jauch KW, et al. Urokinase system expression in gastric carcinoma: prognostic impact in an independent patient series and first evidence of predictive value in preoperative biopsy and intestinal metaplasia specimens. Cancer 2006;106:1026–35. [21] Han SL, Tang HJ, Hua YW, Ji SQ, Lin DX. Expression of COX-2 in stomach cancers and its relation to their biological features. Dig Surg 2003;20:107–14. [22] Vidal O, Metges JP, Elizalde I, Valentini M, Volant A, Molina R, et al. High preoperative serum vascular endothelial growth factor levels predict poor clinical outcome after curative resection of gastric cancer. Br J Surg 2009;96:1443–51.
